Numerous studies have clearly demonstrated the general inability of the humoral immune system in developing functionally effective neutralizing antibodies during natural HIV infections or vaccinations. Further, most of the functional neutralizing antibodies isolated usually possess structures that rarely exist in naturally produced antibodies. Understanding the structural effect of antibody on neutralizing activity is an urgent mission in the practical application of passive immunotherapy. We have found that the IgA1 isotype-switch variant of the CD4i antibody, F425A1g8, displays a significantly increased neutralizing activity, whereas there is little neutralizing activity by any of the IgG subclasses i the absence of sCD4. Moreover, in a small pilot study, CD4i IgA was more effective at reducing viral load in a humanized mouse model than the IgG1 variant of the antibody. While it has clearly been shown that there is an IgA mucosal immune response in HIV-negative sex workers and HIV-status discordant couples, this is not readily observed in HIV infected individuals. Thus, it has been difficult to discern the role of mucosal IgA in preventing or inhibiting HIV infection.It is also known that Fab fragments or scFv molecules of CD4i antibodies are significantly more effective at neutralizing HIV than the intact IgG molecule, likely the result of increased access o the smaller fragments to the epitope on HIV. However, the use of CD4i antibody fragments for therapy is limited by the short half-life of the molecules and scFvs are not normally produced in response to vaccination. The structure of IgA may confer similar access properties of the fragment with a longer half-life and the ability to be elicited as part of the immune response. The CD4i epitope represents a relatively highly conserved epitope integral to viral infection. Therefore, we hypothesize that IgA isotypes of CD4i antibodies through their unique molecular structure can increase the neutralizing activity against HIV infection. Further, we propose that the structure of secretory IgA does not preclude the ability of the more extended IgA1 Fab region from gaining access to the CD4i epitope based on published crystal structures. To test our hypotheses and extend our research on the F425A1g8 IgA, we propose to generate two more IgA variants of CD4i antibodies including E51 and 17b, to confirm the increased neutralizing activity of IgA isotypes of CD4i antibodies. We will not only compare the activity of IgG1 antibodies with IgA1 and igA2 variants, but will also include secretory IgA. Specific functions will be selected based on hypothesized mechanisms active in vivo and include direct viral neutralization; transmission by epithelial cells (as assessed by transcytosis), and participation in effector mediated destruction of HIV (antibody dependent cell-mediated viral inhibition, ADCVI). In addition to functional assays, the stability of the constructs will be assessed under conditions that mimic in vivo conditions. Successful completion of these aims will help to understand the isotype structure-function relation in viral neutralization and directl contributes to HIV vaccine development and the development of passive immunotherapy for prevention and/or treatment of HIV.